A method for producing d-tagatose by a three-enzyme cascade

Abstract

The application discloses a method for producing D-tagatose by a three-enzyme cascade method, and belongs to the field of biotechnology. In the method, fructose is used as a substrate, a reaction system containing metal ions and ATP is used, fructose kinase FRK derived from Clostridium acetobutylicm, D-tagatose 6-phosphate 4-epimerase GatZ derived from Caldilinea aerophila DSM 14535 and D-tagatose-6-phosphate phosphatase MmPase derived from Methanothermobacter marburgensis are used to produce D-tagatose. The yield of D-tagatose is 2.7-9 times of the original yield by optimizing the temperature, the type and the concentration of the metal ions.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for producing D-tagatose by a three-enzyme cascade method, and belongs to the field of biotechnology. BACKGROUND

[0002] Rare sugars are monosaccharides and sugar alcohols that rarely exist in nature. D-tagatose is a C-4 epimer of D-fructose, and is a rare sugar that only exists in a few species in nature. The sweetness of D-tagatose can reach 92% of sucrose, but the energy is only about 30% of the same amount of sucrose, making it an ideal sucrose substitute, and can effectively reduce the incidence of obesity and diabetes, and eliminate oral diseases.

[0003] Currently, the production of D-tagatose in industry is mainly based on D-galactose as a substrate, which is catalyzed by L-arabinose isomerase, but this method has high substrate price, high process cost, and long reaction period. It is urgent to find a method for producing D-tagatose with shortened reaction period, low cost and high product yield. SUMMARY

[0004] The present application provides a method for producing D-tagatose by three-enzyme catalysis from D-fructose, and the product yield is greatly increased by exploring the effects of temperature, metal ion type and concentration. The present application starts from low-cost D-fructose, and synthesizes D-tagatose through phosphorylation and dephosphorylation. In order to improve the yield of D-tagatose synthesized by the method, the effects of temperature, metal ion type and concentration on the synthesis of D-tagatose are explored by experiment, and the final yield of D-tagatose reaches 67.4%.

[0005] The first object of the present application is to provide a method for producing D-tagatose by three-enzyme catalysis, which is based on fructose as a substrate, and utilizes fructokinase (FRK), D-tagatose 6-phosphate 4-epimerase (GatZ) and D-tagatose-6-phosphate phosphatase (MmPase) to produce D-tagatose in a reaction system containing coenzyme and ATP.

[0006] In one embodiment, the FRK is derived from Clostridium acetobutylicm, the GatZ is derived from Caldilinea aerophila DSM 14535, and the MmPase is derived from Methanothermobacter marburgensis.

[0007] In one embodiment, the nucleotide sequence of the gene encoding the FRK is shown in SEQ ID NO. 1, the nucleotide sequence of the gene encoding the GatZ is shown in SEQ ID NO. 2, and the nucleotide sequence of the gene encoding the MmPase is shown in SEQ ID NO. 3.

[0008] In one embodiment, the FRK, GatZ and MmPase are expressed by E. coli.

[0009] In one embodiment, the E. coli is E. coli BL21(DE3).

[0010] In one embodiment, the reaction temperature is 40-60°C.

[0011] In one embodiment, the co-factor is a metal ion.

[0012] In one embodiment, the metal ion is one or more of Mg 2+ , Co 2+ , Mn 2+ , Ni 2+ , Cu 2+ or Al 3+ .

[0013] In one embodiment, the final concentration of the metal ion is 1-4 mM.

[0014] In one embodiment, the reaction time is 8-24 h.

[0015] In one embodiment, the reaction system further comprises a buffer.

[0016] In one embodiment, the buffer is a phosphate buffer.

[0017] A second object of the present application is to provide a method for increasing the yield of D-tagatose, the method comprising adding a co-factor to a reaction system for producing D-tagatose.

[0018] In one embodiment, the co-factor is a metal ion.

[0019] In one embodiment, the metal ion is one or more of Mg 2+ , Co 2+ , Mn 2+ , Ni 2+ , Cu 2+ or Al 3+ .

[0020] In one embodiment, the final concentration of the metal ion is 1-4 mM.

[0021] In one embodiment, the reaction system comprises fructose, ATP, FRK, GatZ and MmPase.

[0022] Advantages:

[0023] This invention provides a one-pot enzymatic method for producing D-tagatose. Using fructose as a substrate, in a reaction system containing metal ions and ATP, the method utilizes fructokinase FRK derived from Clostridium acetobutylicm, D-tagatose 6-phosphate 4-epimerase GatZ derived from Caldilinea aerophila DSM 14535, and D-tagatose-6-phosphate phosphatase MmPase derived from Methanothermobacter marburgensis to produce D-tagatose. By optimizing the reaction conditions (temperature, type and concentration of metal ions), the yield of D-tagatose is increased by 2.7 to 9 times compared to previous methods. The method provided by this invention significantly shortens the reaction cycle compared to existing technologies, requiring only 18 hours, and is low-cost with a high product yield, ultimately achieving a D-tagatose yield of 67.4%. Attached Figure Description

[0024] Figure 1 SDS-PAGE analysis of cell lysate supernatant and precipitate containing fructokinase (FRK), D-tagatose-6-phosphate-4-epimerase (GatZ), and D-tagatose-6-phosphate phosphatase (MmPase). Wherein, M: Blue Plus II Protein Marker; 1: FRK supernatant; 2: FRK precipitate; 3: GatZ supernatant; 4: GatZ precipitate; 5: MmPase supernatant; 6: MmPase precipitate;

[0025] Figure 2 Different Mg 2+ The effect of concentration on the reaction;

[0026] Figure 3 The effect of temperature on the reaction;

[0027] Figure 4 Product yield over time curve;

[0028] Figure 5 The effect of different metal ions on the reaction. Detailed Implementation

[0029] The E. coli BL21(DE3) and pET-28a plasmid involved in the following examples were purchased from Takara Bio Inc., and pG-KJE8 was purchased from Takara Bio Engineering (Dalian) Co., Ltd.

[0030] The fructokinase (FRK) involved in the examples is derived from Clostridium acetobutylicm, the D-tagatose 6-phosphate 4-epimerase (GatZ) is derived from Caldilinea aerophila DSM 14535, the D-tagatose-6-phosphate phosphatase (MmPase) is derived from Methanothermobacter marburgensis.

[0031] The culture media involved in the following examples are as follows:

[0032] LB liquid medium with kanamycin resistance: yeast extract 5.0 g L -1 , peptone 10.0 g L -1 , NaCl 10.0 g L -1 , kanamycin 50 mg L -1 .

[0033] LB solid medium with kanamycin resistance: yeast extract 5.0 g L -1 , peptone 10.0 g L -1 , NaCl 10.0 g L -1 , agar powder 15 g L -1 , kanamycin 50 mg L -1 .

[0034] LB liquid medium with kanamycin and chloramphenicol resistance: yeast extract 5.0 g L -1 , peptone 10.0 g L -1 , NaCl 10.0 g L -1 , kanamycin 50 mg L -1 , chloramphenicol 20 mg L -1 .

[0035] LB solid medium with kanamycin and chloramphenicol resistance: yeast extract 5.0 g L -1 , peptone 10.0 g L -1 , NaCl 10.0 g L -1 , agar powder 15 g L -1 , kanamycin 50 mg L -1 , chloramphenicol 20 mg L -1 .

[0036] The detection methods involved in the following examples are as follows:

[0037] Determination of D-tagatose: high performance liquid chromatography (HPLC) detection: Ca 2+Chromatographic column; mobile phase is pure water, flow rate of mobile phase is 0.9 mL / min; temperature of chromatographic column is 80℃; detector is differential refractive index detector.

[0038] Calculation of yield of D-tagatose product: molar concentration of generated D-tagatose / molar concentration of initial D-fructose*100%.

[0039] Example 1: Construction of recombinant plasmid and recombinant strain

[0040] The FRK gene (Reference Sequence Accession Number: KHD36265.1), the GatZ gene (Reference Sequence Accession Number: WP_014433578), and the MmPase gene (Reference Sequence Accession Number: WP_013296249.1) were obtained from NCBI, and were optimized according to the preference of Escherichia coli codons, to obtain the FRK gene with the nucleotide sequence as shown in SEQ ID NO. 1, the GatZ gene with the nucleotide sequence as shown in SEQ ID NO. 2, and the MmPase gene with the nucleotide sequence as shown in SEQ ID NO. 3. The obtained genes were sent to a company for synthesis of expression plasmids pET28a-FRK, pET28a-GatZ, and pET28a-MmPase.

[0041] The plasmids pET28a-FRK and pET28a-GatZ synthesized by the company were respectively transformed into Escherichia coli BL21(DE3) competent cells, and the transformation products were coated on kanamycin-resistant LB solid culture medium, and cultured at 37℃ for 12-14 h. The transformants were picked on the LB solid culture medium to obtain recombinant Escherichia coli E. coli BL21(DE3) / pET28a-FRK and E. coli BL21(DE3) / pET28a-GatZ containing recombinant plasmids.

[0042] When foreign proteins are expressed in E. coli, the expressed proteins often form inclusion bodies or are degraded by proteases, which is mostly due to the failure of the expressed proteins to fold correctly, which is not conducive to the study of protein function. Molecular chaperones or chaperone proteins are involved in the folding process of proteins. The construction of a co-expression system of a target protein and a chaperone protein can increase the recovery rate of soluble proteins. A protein in the cytoplasm of a molecular chaperone assists the folding, assembly, transport and degradation of biological macromolecules by consuming ATP, thereby greatly improving the soluble expression of the target protein. For example, the chaperone plasmids pG-KJE8, pGRO7 and pG-TF2 containing chaperone proteins GroES, GroEI, DnaK, DanJ, GrpE and trigger factor (TF) can significantly improve the soluble expression of MmPase. The synthetic plasmid pET28a-MmPase of the company was co-transformed into the competent cells of E. coli BL21 (DE3), and the transformation product was coated on LB solid medium with kanamycin and chloramphenicol double resistance, and cultured at 37°C for 12-14h. The transformants were obtained on the LB solid medium to obtain E. coli BL21 (DE3) / pG-KJE8 / pET28a-MmPase containing recombinant plasmids.

[0043] Example 2: Preparation and purification of enzymes

[0044] (I) Protein expression

[0045] The recombinant E. coli BL21 (DE3) / pET28a-FRK, E. coli BL21 (DE3) / pET28a-GatZ and E. coli BL21 (DE3) / pG-KJE8 / pET28a-MmPase obtained in Example 1 were respectively inoculated into LB liquid medium and cultured at 37°C for 10-12h to obtain seed liquids;

[0046] The above seed liquids were respectively inoculated into LB liquid medium at an inoculation amount of 1% (v / v) and cultured at 37°C and 180rpm until the OD 600 was 0.8-0.9. Then, IPTG was added to the culture medium at a final concentration of 0.2mmol / L, and the induction culture was continued at 16°C and 180rpm for 16h to obtain fermentation liquor.

[0047] The fermentation liquor was centrifuged at 4°C and 8000rpm for 10min to collect the bacterial cells. The bacterial cells were broken to obtain cell broken liquid. The cell broken liquid was centrifuged to obtain cell broken liquid supernatant and precipitate. The cell broken liquid supernatant and precipitate were analyzed by SDS-PAGE, and the results are shown inFigure 1 As shown, the recombinant protein was well expressed in soluble form and could be used for subsequent protein purification.

[0048] (II) Protein purification

[0049] The crude enzyme solution (cell lysate supernatant) of fructokinase, D-tagatose 6-phosphate 4-epimerase and D-tagatose-6-phosphate phosphatase obtained in step (1) was subjected to nickel column affinity chromatography purification, respectively, and the specific steps were as follows:

[0050] (1) Preparation of solutions:

[0051] A solution: 0.5 mol / L NaCl, 20 mmol / L imidazole, 25 mmol / L Tris-HCl, 5% glycerol (m / m), pH = 7.4.

[0052] B solution: 0.5 mol / L NaCl, 500 mmol / L imidazole, 25 mmol / L Tris-HCl, 5% glycerol (m / m), pH = 7.4.

[0053] Lysis solution: 0.5 mol / L NaCl, 20 mmol / L PBS, 50 mmol / L EDTA, pH = 7.0.

[0054] NiSO4: 100 mmol / L NiSO4.

[0055] (2) Operation:

[0056] Column regeneration: a 1 mL Ni-NTA pre-loaded gravity column was selected for protein purification. First, 10 times the column volume of A solution was added to the column to clean the column, and when the A solution in the column was almost washed out, 10 times the column volume of ultrapure water was quickly added to rinse, 10 times the column volume of lysis solution was added to clean, and finally 10 times the column volume of Johnson ultrapure water was added to rinse to ensure that the column was fully washed. 10 times the column volume of prepared NiSO4 solution was added to the column, and the column was kept for 5 min. After observing that the column filler showed obvious blue-green color, the NiSO4 solution was discarded and 20 times the column volume of A solution was added to the column to complete the preparation.

[0057] Loading: the supernatant obtained after removing impurities was loaded onto the column, and then 10 times the column volume of A solution was added to the column.

[0058] Elution: different gradient elution methods were used to elute the target protein, and by adjusting the ratio of A and B to set different concentrations of imidazole (50 mmol / L, 100 mmol / L, 150 mmol / L, 200 mmol / L, 250 mmol / L, 300 mmol / L, 350 mmol / L, 400 mmol / L, 500 mmol / L) to elute the target protein adsorbed on the column. The eluate of each gradient was collected.

[0059] Storage: 10 column volumes of B were added to clean the column, followed by 10 column volumes of ultrapure water for flushing, and finally the column was stored with 20% ethanol. The obtained eluate of each gradient was collected with a 10 mL centrifuge tube, and the concentration of the target protein in the eluate was analyzed by SDS-PAGE to determine the appropriate elution concentration of the target protein. The determined single crude band was repeatedly concentrated and replaced at 4°C, 3200 r / min by using 10 kDa or 30 kDa ultrafiltration tubes, and finally the obtained pure enzyme was divided into 1.5 mL centrifuge tubes for subsequent enzymatic property research.

[0060] The pure enzyme solutions containing FRK, GatZ, and MmPase were prepared respectively, and the protein content in the enzyme solution was determined by NanoDrop2000c instrument.

[0061] Example 3: Effect of metal ion concentration on cascade reaction

[0062] The reaction system involved in the example was 10 mM fructose, 10 mM ATP, 0.2 mg / ml FRK, 0.5 mg / ml GatZ, 0.3 mg / ml MmPase, and the buffer was PBS (50 mM, pH 7.5), and 1 mM, 2 mM, 3 mM, and 4 mM of MgCl2·6H2O were added respectively. The control was the reaction system without adding metal ions.

[0063] The reaction was carried out at 40°C, and samples were taken at different times for high performance liquid chromatography analysis, and the results are shown in Figure 2 and Table 1.

[0064] Table 1 Different Mg 2+ concentrations

[0065] Concentration (mM) Reaction time (h) Maximum yield of product (%) Control 12 6.1 1 12 19.6 2 12 39.4 3 12 48.1 4 12 49.3

[0066] From Figure 2 and Table 1, when the Mg 2+ concentration is higher than 3 mM (including 3 mM), the reaction can reach a high product yield in 12 h, and the product yield is close to 50%, combined with the separation and purification of the product, the metal ion concentration of 3 mM is selected as the basis for further research.

[0067] Example 4: The effect of temperature on cascade reactions

[0068] The reaction system involved in the examples consisted of a final concentration of 10 mM fructose, 10 mM ATP, 0.2 mg / ml FRK, 0.5 mg / ml GatZ, and 0.3 mg / ml MmPase. The buffer was PBS (50 mM, pH 7.5), with 3 mM Mg added. 2+ .

[0069] The reactions were carried out at 40℃, 45℃, 50℃, 55℃, and 60℃, respectively. Samples were taken at different times and analyzed by high-performance liquid chromatography. The results are shown in the figure. Figure 3 As shown in Table 2.

[0070] Table 2 Reactions at different temperatures

[0071] Temperature Reaction time (h) Maximum yield of product (%) 40℃ 12 46.0 45℃ 24 47.6 50℃ 8 19.5 55℃ 4 5.0 60℃ 8 3.8

[0072] Depend on Figure 3 As shown in Table 2, the product yield is higher at 40℃, reaching 46.0% after 12 hours of reaction. Therefore, 40℃ was chosen as the basis for subsequent research.

[0073] Example 5: Product yield curve over time

[0074] The reaction system involved in the examples consisted of a final concentration of 10 mM fructose, 10 mM ATP, 0.2 mg / ml FRK, 0.5 mg / ml GatZ, and 0.3 mg / ml MmPase. The buffer was PBS (50 mM, pH 7.5), with 3 mM Mg added. 2+ .

[0075] The reaction was carried out at 40℃, and samples were taken at different times for high-performance liquid chromatography analysis. The results are shown in the figure. Figure 4 As shown in Table 3.

[0076] Table 3 Time-series response

[0077]

[0078] Depend on Figure 4 As shown in Table 3, the reaction reaches equilibrium at 18 hours, and the product yield reaches its maximum value of 51.5%. Sampling and testing at 18 hours of reaction will serve as the basis for subsequent research.

[0079] Example 6: Effects of different metal ions on cascade reactions

[0080] The reaction system involved in the examples consisted of a final concentration of 10 mM fructose, 10 mM ATP, 0.2 mg / ml FRK, 0.5 mg / ml GatZ, and 0.3 mg / ml MmPase. The buffer was PBS (50 mM, pH 7.5), with different metal ions added to a final concentration of 3 mM. The control group consisted of a reaction system without added metal ions.

[0081] The reaction was carried out at 40℃, and samples were taken after 18 hours for high-performance liquid chromatography analysis. The results are shown below. Figure 4 As shown in Table 3.

[0082] Table 4 Reactions of different metal ions

[0083] Metal ion Maximum yield of product (%) Control 7.5 Mg 2+ ]]> 48.8 Co 2+ ]]> 67.4 Al 3+ ]]> 56.2 Mn 2+ ]]> 48.7 Ni 2+ ]]> 20.7 Cu 2+ ]]> 2.4

[0084] Depend on Figure 5 As shown in Table 4, the addition of Co... 2+ Al 3+ Mn 2+ Ni 2+ and Mg 2+ The product yields of the reactions were all higher than those without the addition of metal ions, with the addition of Co being particularly significant. 2+ It can increase the product yield up to 9 times the original.

[0085] Comparative Example 1

[0086] Recombinant *E. coli* were obtained using the method described in Example 1, consisting of D-tagatose-6-phosphate phosphatase GatZ (Reference Sequence No.: WP_014433578) from *Caldilinea aerophila* DSM 14535 and D-tagatose-6-phosphate phosphatase T6PP (Reference Sequence No.: ACM23254.1) from *Thermotoga neapolitana* DSM 4359. Pure enzyme solutions were then obtained according to the method described in Example 2. The pure enzyme solutions were subjected to a cascade reaction. The reaction system consisted of a final concentration of 10 mM fructose, 10 mM ATP, 0.2 mg / ml FRK, 1 mg / ml GatZ, 0.2 mg / ml T6PP, and 2 mM magnesium ions. The buffer was PBS (50 mM, pH 10). 7.5) The reaction was carried out at 50℃. Samples were taken at 2h, 4h, 12h and 24h of the reaction and analyzed by high performance liquid chromatography. The results showed that no product was detected.

[0087] Comparative Example 2

[0088] D-tagatose 6-phosphate 4-epimerase FbaA (Reference Sequence accession number: EFE61766.1) derived from Escherichia coli B088 and phytase Phytase purchased from Shanghai Yuye Company were used to obtain recombinant E. coli according to the method of Example 1, and pure enzyme solution was obtained according to the method of Example 2. The obtained pure enzyme solution was subjected to cascade reaction. The reaction system was 10 mM fructose, 10 mM ATP, 0.2 mg / ml FRK, 0.5 mg / ml FbaA, 5 mg / ml Phytase, and the buffer was PBS (50 mM, pH 7.5). The reaction was carried out at 50°C. Samples were taken at 2h, 4h, 12h and 24h, and high performance liquid chromatography analysis was carried out. The results showed that no product was detected.

[0089] Comparative Example 3

[0090] D-tagatose 6-phosphate 4-epimerase GatZ (Reference Sequence accession number: WP_014433578) derived from Caldilinea aerophila DSM 14535 and phytase Phytase purchased from Shanghai Yuye Company were used to obtain recombinant E. coli according to the method of Example 1, and pure enzyme solution was obtained according to the method of Example 2. The obtained pure enzyme solution was subjected to cascade reaction. The reaction system was 10 mM fructose, 10 mM ATP, 0.8 mg / ml FRK, 0.2 mg / ml GatZ, 5 mg / ml Phytase, 2 mM magnesium ion, and the buffer was PBS (50 mM, pH 7.5). The reaction was carried out at 50°C. Samples were taken at 2h, 4h, 12h and 24h, and high performance liquid chromatography analysis was carried out. The results showed that the yield of D-tagatose was only 5.1% at 24h.

[0091] Although the present application has been disclosed with reference to the preferred embodiments above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application. Therefore, the protection scope of the present application should be defined by the claims.

Claims

1. A method for the production of D-tagatose catalyzed by three enzymes, characterized in that, The method is to produce D-tagatose by using fructose kinase FRK, D-tagatose 6-phosphate 4-epimerase GatZ and D-tagatose-6-phosphate phosphatase MmPase in a reaction system containing metal ions and ATP with fructose as a substrate; The FRK is derived from Clostridium acetobutylicm, the GatZ is derived from Caldilinea aerophila DSM 14535, and the MmPase is derived from Methanothermobacter marburgensis; the nucleotide sequence of the gene encoding the FRK is shown as SEQ ID NO. 1, the nucleotide sequence of the gene encoding the GatZ is shown as SEQ ID NO. 2, and the nucleotide sequence of the gene encoding the MmPase is shown as SEQ ID NO. 3; The metal ion is Al 3+ The final concentration of the metal ion is 3-4 mM; the reaction temperature is 40-45°C, and the reaction time is 12-24 h.

2. The method of claim 1, wherein, The FRK, the GatZ and the MmPase are expressed by using E. coli.

3. The method of claim 2, wherein, The E. coli is E. coli BL21 (DE3).

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